The Vitamin D receptor (VDR) is a nuclear receptor important for regulating calcium absorption. Humans with mutations in the VDR develop Hereditary Vitamin D Resistant Rickets. In addition to Rickets, multiple inherited VDR mutations result in alopecia totalis. VDR null mice phenocopy the human disease. While the rachitic changes in the mice can be rescued by diet, alopecia remains, suggesting a unique role for the VDR in the skin. Previous studies demonstrated that this is due to loss of ligand-independent functions of the VDR in the hair follicle resulting in decreased keratinocyte stem cell (KSC) number. In addition, there is a defect in KSC function manifested by an inability to regenerate hair follicles when KSC numbers are not decreased. VDR ablation results in disruption of canonical Wnt/[unreadable]-catenin signaling in primary keratinocytes. Furthermore, the VDR was found to co-immunoprecipitate with [unreadable]-catenin and Lef1. Therefore, we hypothesize that the VDR activates Wnt-dependent hair follicle regeneration through a functional interaction with components of the Wnt signaling cascade. Using a GST-fusion based approach, we have identified a novel interaction between the unliganded VDR and Lef1 that does not require [unreadable]-catenin. This proposal will further characterize the interaction between the VDR and Lef1. In Specific Aim I, the precise domains of the VDR and Lef1 necessary and sufficient for interaction will be identified using PCR-based site directed mutagenesis followed by protein purification and studies of protein interaction. In addition, purified recombinant proteins will be generated to determine whether the interaction between the VDR and Lef1 is direct or if additional nuclear factors are required. Specific Aim II will establish the functionality of the VDR/Lef1 interaction. Transient transfection of primary keratinocytes lacking the VDR will be used to determine if the interaction between the VDR and Lef1 is essential for activation of a Wnt reporter gene. Through characterizing this novel interaction, we will determine how the unliganded VDR contributes to regulation of hair follicle regeneration. These studies will not only determine a new role for the unliganded VDR, but may also identify therapeutic targets for treatment of alopecia. These studies will determine the molecular mechanisms by which the VDR regulates hair follicle regeneration by characterizing the interaction between the VDR and Lef1. Canonical Wnt signaling is essential for multiple biological processes, therefore understanding how the VDR regulates Wnt dependent hair follicle regeneration may have implication for understanding related processes such as wound healing.